Hum. Reprod. Advance Access originally published online on January 12, 2006
Human Reproduction 2006 21(5):1172-1178; doi:10.1093/humrep/dei484
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Evidence of a high proportion of premature unbalanced separation of sister chromatids in the first polar bodies of women of advanced age
1 Department of reproductive biology, cytogenetics, gynaecology and obstetrics, CHI Poissy-Saint Germain, Poissy, 2 Institut National de la Santé et de la Recherche Médicale (INSERM U 407), Faculté de Médecine Lyon-Sud, Oullins, 3 Department of reproductive biology and development, CHU de Nancy, Maternité A Pinard, Nancy, 4 Department of genetics & reproductive biology, Laboraroire Lefaure & Petit, Epinal and 5 EA 3442 "Génétique, Signalisation, Différenciation" Université Henri Poincaré, Nancy, France
6 To whom correspondence should be addressed at: Department of reproductive biology, cytogenetics, gynaecology and obstetrics, CHI Poissy-Saint Germain, 10 rue du Champ Gaillard, 78303 Poissy cedex, France. E-mail: fvialard{at}hotmail.com
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Abstract |
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BACKGROUND: Maternal ageing is the only aetiological factor unequivocally linked to aneuploidy. Two mechanisms seem to explain these abnormalities in oocytes: non-disjunction and premature unbalanced separation of sister chromatids (PSSC). Previous studies of unfertilized oocytes argue for a major role of PSSC in the aetiology of aneuploidy for women of advanced age, but in vitro ageing of the oocytes could influence the results. METHODS: Owing to the high prevalence of aneuploidy in women of advanced age, chromosomal screening of the first polar body just before ICSI was offered to women (from 38 years of age) included in an assisted reproduction programme. RESULTS: Among 141 oocytes from 29 women (mean age 40 years and 2 months), 43 (30.5%) were abnormal. Sixty-five abnormalities were found and PSSC was involved in 80% of cases. CONCLUSION: These results are in accordance with previous studies and confirm, in ‘fresh’ oocytes, the major role of PSSC in the aetiology of aneuploidy in women of advanced age.
Key words: aneuploidy/age/premature separation of sister chromatids/cohesin/polar body
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Introduction |
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More than 70% of aneuploidies result from female meiosis and occur during the first meiotic division (Pellestor, 1991
; Hassold and Hunt, 2001). Hassold et al. (1996) have demonstrated the maternal origin of the most frequent fetal trisomies, i.e. 93% of trisomy in 18, 95% in 21 and 100% in 16. Maternal ageing is the only aetiological factor unequivocally linked to aneuploidy, and a recent study provides direct evidence of this link in unfertilized oocytes (Pellestor et al., 2003). Two mechanisms seem to explain these abnormalities in oocytes. The first is non-disjunction of a whole chromosome during meiosis. The second is premature separation of sister chromatids, which can be balanced or unbalanced. This unbalanced situation [premature unbalanced separation of sister chromatids (PSSC)] results in an extra or a missing oocyte copy of a single chromatid. Pellestor et al. (2002) recently analysed a series of 1397 karyotypes of unfertilized oocytes after IVF attempts (women aged 19–46 years) and showed that PSSC was more frequent than non-disjunction. Moreover, PSSC seems to be more influenced by maternal ageing than malsegregation (Pellestor et al., 2003). This hypothesis has been discussed since 1991 when Angell (1991) first hypothesized that this phenomenon was the main source of human aneuploidy.
These PSSC evaluations were obtained by analysing mainly unfertilized IVF oocytes with a poor technical success rate (less than 50%). A possible selection of abnormal oocytes during fertilization can be discussed as well as a possible influence of oocyte in vitro ageing on the technical results. Only one recent paper deals with the first and second polar bodies (PBs), simultaneously biopsied and analysed in fertilized oocytes from older patients, and confirms a very high rate of PSSC (Kuliev et al., 2003). In the latter study, the first PB was aged 20 h in vitro before biopsy. No evidence has yet been found for a high proportion of PSSC in fresh oocytes from older patients. Only the first PB diagnosis is able to give such information, because the biopsy is performed on oocytes retrieved just before fertilization. This approach may avoid artefactual PSSC owing to metaphase II oocytes ageing in culture and enable evaluation of the proportion of the two mechanisms underlying oocyte aneuploidy.
Nevertheless, segregation of PSSC (Figure 1) in theory is most likely random, leading to embryonic aneuploidy in 50% of cases unlike non-disjunction where 100% of resulting embryos are aneuploid. On the other hand, abnormalities as well as meiotic corrections may occur later after fertilization and this may explain the high rate of mosaicism in preimplantation embryos (Harper et al., 1995; Delhanty et al., 1997; Laverge et al., 1997; Munne et al., 1998; Los et al., 2004; Daphnis et al., 2005). Despite these limitations of pre-embryo or oocyte diagnosis, and in order to prevent the high rate of aneuploidy in embryos from women aged 35–38 years or more, several teams from different countries use preimplantation genetic screening (PGS) and/or preconceptional screening (PCS) for these patients. The development of fluorescence in-situ hybridization (FISH) techniques allowed the PGS for chromosomal abnormalities, and the first studies confirmed the expected high rate of chromosome abnormalities among embryos from older women (Verlinsky et al., 1999; Kahraman et al., 2000; Gianaroli et al., 2002; Pehlivan et al., 2003). The abnormality rate reported in these studies depends on the number of chromosomes tested and was close to 40% for a set of five probes (Kahraman et al., 2000) and close to 70% for an eight-probe set (Gianaroli et al., 2002) for women over 35 years of age. The most comprehensive study of embryo chromosomes was achieved using comparative genomic hybridization and indicated that 75% of embryos contain at least one aneuploid cell (Voullaire et al., 2000; Wells and Delhanty, 2000). Unlike preimplantation diagnosis which is not accepted by the French bioethics law for older women, PCS can be proposed before ICSI in France as a research protocol. No deleterious effect on embryo development has yet been reported after PB biopsy (Lee and Munne, 2000).
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We used first PB biopsy and FISH diagnosis in order to select oocytes before spermatozoa microinjection and to evaluate the incidence of the two mechanisms involved in aneuploidy: non-disjunction and PSSC. We report here our clinical results after 29 trials leading to the screening of 141 oocytes, 5 pregnancies and 4 births. The chromosome results of the first PB analysis from two French fertility centres are analysed. They confirm the high proportion of PSSC in ‘fresh’ first PBs from older patients.
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Materials and methods |
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Patients
First PB analysis before ICSI was proposed to women over 38 years of age. This age limit is considered as an indication of prenatal diagnosis for advanced maternal age in France. Our PB evaluation protocol was accepted by our local ethics committee and by the French health authorities. Informed consent was given following genetic counselling. This protocol was proposed in two French IVF centres (Nancy and Poissy).
A total of 29 women were included for one preconception diagnosis. Multiple follicular growth was induced by exogenous gonadotrophins following a desensitization protocol with GnRH analogues according to a long down-regulation protocol (17 cycles) or a short protocol (12 cycles). Oocytes were retrieved approximately 36 h after HCG administration.
Table I summarizes the population characteristics with a mean maternal age of 40 years 2 months (range: 38–42). Infertility was primary for 22 patients and secondary for 7. The mean number of previous IVF attempts was 2.8. ICSI was performed for severe male infertility (14 cycles) or previous IVF failure (15 cycles).
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Polar body biopsy
PB biopsy was carried out using the Zilos TK laser (Hamilton Thorne Biosciences). Immediately after oocyte retrieval and decoronization, three or four laser impacts (180 mW, 0.5 s pulse) were made in the zona pellucida. The PB was then extracted (165 oocytes) with a biopsy micropipette (Humagen, Charlottesville, VA, USA) (Figure 2) and placed in 0.5 µl of water on a siliconized slide. This water drop was air dried and two drops of Carnoy solution were added.
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FISH procedure
The slide was then transferred into different solutions: 5 min in methyl alcohol at room temperature (RT), 10 min in 2xsaline sodium citrate (SSC) at 37°C, 10 min in a 1% paraformaldehyde solution at RT, 5 min in PBS at RT, 10 min in a 0.1 N HCl solution with 30 µl/40 ml of 10% active pepsin solution, 5 min in PBS at RT and 2 min in 70, 85 and 100% ethyl alcohol for dehydration.
A 3 µl drop of probe solution was placed on each PB. Co-denaturation and hybridization were automatically performed in the Hybrite (Vysis) (73°C for 4 min and 37°C for 4 h).
The slide was then washed in two solutions: 1 min 45 s in a 0.7xSSC/0.3% NP40 solution, 15 s in a 2xSSC/0.1% NP40 solution, and counterstained with an antifade solution and analysed with a five-filter fluorescent microscope (Olympus BX60) and the Pathvysion imaging system (Digital Scientific) (Figure 3).
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PBs were analysed by one of two different probe mixtures: the first hybridization mixture (Poissy group: panel 1) was MultivysionTM Polar Body Kit probe panel hybridization mixture marketed by Abbott including LSI® 13 (13q14) labelled with SpectrumRedTM, CEP® 16 (satellite II D16Z3) with SpectrumAquaTM, CEP® 18 (alpha satellite D18Z1) with SpectrumBlueTM, LSI® 21 (21q22.13–21q2.2) with SpectrumGreenTM and LSI® 22 (22q11.2) with SpectrumGoldsTM (Figure 3). The second hybridization mixture (Nancy group: panel 2), included two hybridization steps with centromeric probes (Cytcocell Ltd, Cambridge, UK) for chromosomes 13–21 (alpha satellite D13Z1-D21-Z1), 14–22 (alpha satellite D14Z1-D22-Z1), 16 (alpha satellite D16Z1) and 18 (alpha satellite D18Z1) labelled, respectively, in SpectrumGreenTM, SpectrumRedTM, SpectrumGreenTM and SpectrumRedTM, and ranging in concentration between 5 and 20 ng/assay. Owing to the cross-hybridization obtained with the second panel, no specific chromosome identification was possible and only results from the first panel were used to collect information about specific chromosome abnormalities (71 oocytes).
Scoring criteria
Each PB chromosome normally consists of two chromatids. With panel 1, locus-specific probes always give a doublet signal corresponding to each chromatid (Figure 3) and each chromosome was represented by a doublet of distinct colour, sometimes very close, or by two separate signals. Centromeric probes usually give large signals for one chromosome or a doublet with very close signals. We have chosen to present every loss or gain of a whole chromosome (two spots) or of one chromatid (one spot). Signal absence or four signals were interpreted as non-disjunction (Figure 4) and one or three signals as PSSC (Figures 5a and b). Of course, the absence of signals may be influenced by artefactual loss during fixation or FISH. This was checked by comparing the rates of abnormal loss or gains of chromosomes or chromatids. Two separate signals were not considered as an abnormality but as a balanced separation of sister chromatids. They were not included in the count of PSSC. With panel 2, the interpretation was the same but four spots (four chromatids) were considered normal for centromeric acrocentric chromosomes, corresponding to two chromosomes (13/21 or 14/22) because of cross-hybridization.
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ICSI procedure
Oocytes without FISH analysis because of technical failure or immaturity (the biopsy was impossible in the morning but the PB was expelled at the time of ICSI in the afternoon) and oocytes with normal FISH results were then microinjected. The opening in the zona pellucida was kept at 12 or 6 o’clock in order to keep the spindle away from the microinjection location. Oocytes were then microinjected with a 7 µm outer diameter ICSI micropipette (Humagen). If necessary, a second partial microdissection was performed in the zona pellucida at the point of microinjection in order to reduce the pressure inside the oocyte and to reduce the risk of extrusion of ooplasm.
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Results |
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The assisted reproduction results are summarized in Table II. Of 184 oocytes showing the first PB (PB1) that were retrieved [mean 6.3 per cycle (3–11)], 89.7% (165) could be biopsied and of these 85.5% (141) were analysed by FISH (76.6% of the matured oocytes). The success rate was higher (P < 0.02) for panel 2 FISH (93.3%; 70/75)) than for panel 1 (78.9%; 71/90). Four oocytes were lysed. Spermatozoa were injected into 141 oocytes, 98 with normal diagnosis by FISH and 43 without diagnosis (24 because of FISH failure, 19 because they could not be biopsied in the morning due to their immaturity [absence of PB (n = 4) or cytoplasmic bridge between the oocyte and PB1 (n = 15)]. Fertilization rate was 64.5% and cleavage rate 93.5%. Pregnancy rate was 17.2% per cycle or 20.8% per transfer, with four healthy infants being born and one spontaneous abortion.
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FISH results are summarized in Table III. Of 141 PB1 analysed, 98 were normal with a partial (5%) or complete (64.5%) result (all signals interpreted), 43 were abnormal (30.5%), 27/71 (38%) with panel 1 (Vysis, Poissy) and 16/70 (23%) with panel 2 (Cytocell, Nancy), with no significant difference between these figures. Among the 43 abnormal PB1, 65 abnormalities were noted, 1.51 per PB (1–3), with 13 (20%) non-disjunction and 52 (80%) sister chromatid predivisions and the mechanism frequencies were not different between the two panels. When the population was divided into two groups according to age (38–39 versus 40 and over), the PSSC rate was, respectively, at 73.8% (31/42) and 91.3% (21/23) (this increase was not statistically significant). Loss (53.9%) and gain (46.1%) of chromosomes or chromatids were equally frequent. Nineteen oocytes were abnormal for only one chromatid (nine loss and eight gain) among 38 oocytes with a sister chromatid unbalanced predivision. The abnormality rates varied greatly from 0 to all oocytes.
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Panel 1 allowed a specific chromosome analysis (71 oocytes were analysed and 41 FISH abnormalities were found) and showed that 9.9% of the oocytes were abnormal for chromosome 13, 5.6% for 16, 5.6% for 18, 19.7% for 21 and 14% for 22, with no difference in abnormality mechanisms. Only the incidence of chromosome 16 and 18 aneuploidies were different from chromosome 21 aneuploidy (P < 0.05).
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Discussion |
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The pregnancy rate was 17.2% per transfer, which is a good result, given the advanced age of the women, but a little lower than that observed in previous PCS reports (21.9%, Kuliev et al., 2003
; 22.5%, Montag et al., 2004) studying younger women (i.e. mean 38.5 years old for Montag et al., 2004) and with a higher number of oocytes retrieved per patient. The fertilization rate (64.5%) and cleavage rate (93.4%) obtained agreed with the data available on the French National Register on in vitro fertilization (http://perso.wanadoo.fr/fivnat.fr/), which suggests that PB1 biopsy had no deleterious effect.
The number of ‘normal’ oocytes per patient was very variable (0–4), the ratio of abnormalities among oocytes ranged from 0 to 100%, and this finding can be of value in predicting the success rate of future assisted reproduction. Unlike PGS, the evaluation of the incidence of aneuploidy was based on the total population of oocytes and not only on the fertilized ones. A few weeks after the attempt, all patients were offered genetic counselling in which the results were summarized and discussed. This approach has also been used after blastomere PGS (Ferraretti et al., 2004).
First PB screening is the only possible preimplantation, especially preconception screening technique for older patients, which is allowed by French legislation. In France, genetic blastomere analysis is only possible for patients with a diagnosed genetic abnormality in the parents, and PGS is prohibited in cases of maternal ageing. First PB screening, which concerns the gamete before fertilization, seems to be a good alternative. It allows analysis of the complete cohort of oocytes (fertilized or not) without in vitro ageing prior to biopsy. We used a laser-assisted procedure, described only once for human oocytes (Montag et al., 2004) and already described for PGD of embryos, because the high precision of the laser beam facilitates zona pellucida microdissection and PB biopsy. Biopsy is sometimes impossible because of a cytoplasmic bridge between the PB and the oocyte due to immaturity or because PB was expelled after biopsy (in the morning) but before ICSI (in the afternoon). All the mature oocytes examined at the time of ICSI, even undiagnosed oocytes, were microinjected because of the small number of oocytes recovered from older women. In our series, only 76.6% of the PBs could be analysed by FISH, but the procedure performed on a single, very small cell with no second chance possible is technically difficult, and the success rate is in accordance with previous reports (81.6%, Verlinsky et al., 1999; 82%, Pujol et al., 2003; 87.2%, Montag et al., 2004); and this procedure is anyway much better than the success rate of karyotyping unfertilized oocytes (45.9%) (Pellestor et al., 2002).
The FISH success rate was higher (78.9 versus 93.3%) when using panel 2 centromeric probes. The spots obtained were larger and FISH screening of PBs on slides was easier because of DAPI counterstaining, which was impossible for panel 1 with the use of CEP® 18 labelled with SpectrumBlueTM. However, only panel 1 permits precise identification of each chromosome and a separate analysis of each chromosome aneuploidy rate.
The aneuploidy rate of PBs was 30.5% in our series, which is lower than that in other series, ranging from 35.7% for PB1 analysis with a three-probe set (Verlinsky et al., 1999) to 40—64% for blastomere PGS (Gianaroli et al., 1999; Kahraman et al., 2000) with a five- to seven-probe set. This figure can be explained taking into account that the first PB approach explores only meiosis I. There is no difference in abnormality rate, but in theory, with panel 2, we may misdiagnose a double compensated error dealing with acrocentric chromosome (13/21 or 14/22) cross-hybridization. We never encountered this situation in the panel 1 results, so this situation is probably very rare.
We excluded oocytes with only one PSSC and only a 50% abnormality risk for the embryo. These oocytes were not numerous in our series, 50% (19/38) of oocytes with one PSSC also showed another abnormality. Hence, the risk of inappropriate exclusion of oocytes can be estimated as 7% (10/141). Anyway, we think we cannot transfer an embryo with a known 50% viable aneuploidy probability evaluated after PCS. This is a general problem in gamete or preimplantation screening. For example, embryos exhibiting three pronuclei (PN) are usually excluded from IVF programmes, but 25% of cleaved, initially 3 PN, embryos are diploid (Bergere et al., 1995; Grossmann et al., 1997) and some of them should in fact be normal. Mosaicisms are also frequent in morphologically normal embryos, thus explaining false-negative (1.5%) and false-positive (5.8%) PGS aneuploidy results (Munne et al., 2002). Furthermore, chromosome normal cell lines occur in a significant proportion of chromosomally abnormal embryos (Munne et al., 2005). The instability of chromosome content in the first embryo mitoses (Harper et al., 1995; Delhanty et al., 1997; Laverge et al., 1997; Munne et al., 1998; Daphnis et al., 2005) makes gamete and embryo aneuploidy screening difficult.
Chromosome or chromatid gain (46%) and loss (54%) were equally frequent, which may suggest that artefactual loss of chromosomes or chromatids was rare when analysing fresh oocytes. But in order to confirm these results, abnormal oocyte controls are needed. The success rate in FISH of control oocytes was not good enough to perform a control in our study. Few studies have reported such evaluation (Cupisti et al., 2003; Pujol et al., 2003) with 75% agreement in analysis of both the PB and the unfertilized oocyte.
Using only panel 1 probes (41 FISH abnormalities), we observed that only 5.6% of oocytes were abnormal for chromosome 16 and 20% for chromosome 21, which is a statistically different (P < 0.05) frequency, a surprising figure compared with the high proportion of chromosome 16 abnormalities in miscarriages (Warburton and Kinney, 1996; Nicolaidis and Petersen, 1998; Hassold and Hunt, 2001) or in unfertilized oocytes of younger women (mean age 33.6) (Pellestor et al., 2002). These discrepancies might be linked to the age of the patients in our study.
First PB analysis can also give information about the mechanism involved in the development of aneuploidy. As previously mentioned, two different mechanisms may be involved, including chromosome malsegregation and PSSC. Angell (1991) first described this phenomenon in oocytes, usually observed in chromosome instability syndromes and in malignancies.(Angell 1991, 1995, 1997) identified sister chromatid separation as the major cause of human oocyte aneuploidy. Several cytogenetic studies have confirmed the coexistence of the two phenomena (Nakaoka et al., 1998; Mahmood et al., 2000). Balanced separation of chromatids in oocytes happens when oocytes have aged in vitro (Munne et al., 1995) and increases with culture time from 6% in fresh PBs to 53% in PBs from eggs cultured for 24–48 h (Munne et al., 1995). Balanced chromatid predivisions might induce artefactual chromatids loss during PB1 fixation after in vitro ageing.
In ‘fresh’ oocytes, 80% of 65 abnormalities were PSSC. These data are similar to those of Pellestor et al. (2002), who found 71% PSSC in unfertilized IVF oocytes, and of Verlinsky et al. (1999) (70% of PSSC) in fertilized oocytes (women who are 35 years and over). Recently, analysing the first PB of unfertilized IVF oocytes with comparative genomic hybridization, PSSC was demonstrated to be involved in two-thirds of the aneuploidy events (women age: 27–42 years) (Gutierrez-Mateo et al., 2004). The factor that might explain the high rate of PSSC in our series of fresh PB1 is the advanced age of the women (mean 40 years 2 months). Pellestor et al. (2003) demonstrated by analysing human unfertilized oocytes that single-chromatid malsegregation is an essential factor in the age-dependent occurrence of non-disjunction. In our series, when the population is divided into two groups, PSSC does not significantly increase but varies from 73.8% (31/42) for women 38–39 years old to 91.3% (21/23) for women of 40 years and over.
Different mechanisms may be involved in sister chromatid cohesion. Koehler et al. (1996) first showed that the occurrence of proximal exchanges might lead to PSSC. Alternatively, achiasmate figures, with no exchange between the two homologous chromosomes, which can line up individually along the metaphase I plate, may result in PSSC, one single chromatid going separately to each pole. Furthermore, a direct correlation has been demonstrated between maternal ageing and specific pathological recombination (Lamb et al., 1997, 2005). Age-dependent deterioration of cellular factors required for proper chromosome exchanges, spindle formation or chromosome cohesion has also been hypothesized (Hawley et al., 1994).
Sister chromatid cohesion is maintained by a particular class of proteins, the cohesins (Michaelis et al., 1997). These proteins contain four subunits: two structural maintenance of chromosome (SMC) proteins—SMC1
(Revenkova et al., 2001) and SMC3 (Klein et al., 1999) and two non-SMC subunits REC8 (Parisi et al., 1999) and STAG3 (Prieto et al., 2001). During the metaphase/anaphase transition, cohesins are cleaved and initiate chromosome segregation (Waizenegger et al., 2000), and their age-related deterioration might explain a gradual loss of chromosome cohesion with maternal age (Wolstenholme and Angell, 2000). The observation of mutant REC8 Schizosaccharomyces pompe which lack sister chromatid cohesion (Molnar et al., 1995), and sterile SMC1 (Revenkova et al., 2004) and REC8 (Xu et al., 2005) deficient mice, with abnormal synapsis, also supports this hypothesis.
Another hypothesis is the relationship between alphoid DNA size reduction and meiosis I abnormalities, especially dealing with chromosome 21 (Marzais et al., 1999; Maratou et al., 2000). This reduction could affect centromere-associated proteins and alter sister chromatid cohesion. Moreover, female meiosis checkpoint control at the metaphase/anaphase transition is less efficient than the male one (LeMaire-Adkins et al., 1997) and the proteins involved in this checkpoint show an age-related degradation of their mRNA (Steuerwald et al., 2001).
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Conclusion |
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In France, first PB screening seems to be a good alternative to PGS since French bioethics legislation excludes women of advanced age from having PGS.
This approach should also help to understand the mechanisms underlying human aneuploidy. Our results were obtained by analysing fresh oocytes with no in vitro ageing and suggest that PSSC is indeed the major cause of chromosomal aneuploidy linked to maternal age, and that G chromosome group aneuploidies are probably the most frequent ones.
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References |
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Submitted on April 22, 2005; resubmitted on December 2, 2005; accepted on December 9, 2005.
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